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G Kapatos

Publications and source records attributed to G Kapatos.

At least 19 recordsLinked to original sources
PubMed1998

Nigrostriatal dopamine neurons express low levels of GTP cyclohydrolase I protein.

A previous study using the in situ hybridization technique showed that serotonin neurons contain substantially more GTP cyclohydrolase I mRNA than do either dopamine or norepinephrine neurons. The objective of the current study was to determine whether these differences in mRNA abundance are predictive of the amount of GTP cyclohydrolase I protein available for tetrahydrobiopterin biosynthesis. The double-label immunofluorescence technique was used to localize GTP cyclohydrolase I protein to the tyrosine hydroxylase-positive A9 dopamine neurons of the substantia nigra and the A6 norepinephrine neurons of the locus ceruleus or the tryptophan hydroxylase-positive B6/B7 serotonin neurons of the dorsal raphe nucleus. Although GTP cyclohydrolase I immunofluorescence within serotonin and norepinephrine neurons was relatively intense, the fluorescence signal within dopamine neurons was faint to nondetectable. An immunoautoradiographic technique was developed to quantify these apparent differences in GTP cyclohydrolase I protein expression at the cellular level. Significant differences between all three neurochemical subdivisions were found and comparisons showed that on average serotonin neurons contain between 2.3- and 7.3-fold more GTP cyclohydrolase I protein than do either norepinephrine or dopamine neurons, respectively. Nigrostriatal dopamine neurons thus appear to synthesize and maintain tetrahydrobiopterin at low levels. Because dopamine and norepinephrine neurons express essentially equal amounts of GTP cyclohydrolase I mRNA, posttranscriptional events may serve to maintain low levels of GTP cyclohydrolase I protein within dopamine neurons. Phenotypic differences in GTP cyclohydrolase I protein expression across populations of monoamine neurons may be an important control point in neurotransmitter biosynthesis.

Animals↗
PubMed1996

Tetrahydrobiopterin biosynthesis in C6 glioma cells: induction of GTP cyclohydrolase I gene expression by lipopolysaccharide and cytokine treatment.

The possibility that 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis is stimulated in glial cells by treatment with lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) was examined in the astrocyte-derived C6 glioma cell line. Under basal culture conditions BH4 levels were found to be at the limit of detection. Concurrent treatment with 10 micrograms/ml LPS and 50 ng/ml TNF-alpha caused a time-dependent 13-fold increase in the levels of BH4. This treatment paradigm also induced nitric oxide synthase activity, as evidenced by increased levels of nitrite, an oxidized metabolite of NO, in the culture medium. LPS and TNF-alpha treatment led to a 25-fold increase in GTPCH enzyme activity, the first and rate-limiting enzyme in BH4 synthesis, and a corresponding 23-fold increase in GTPCH protein levels. Northern blot analysis showed that increased levels of GTPCH mRNA preceded changes in GTPCH protein, GTPCH enzyme activity and BH4 levels and reached a maximal of 44-fold that was sustained for at least 48 h. These results demonstrate that LPS and TNF-alpha stimulate de-novo BH4 biosynthesis and suggest that C6 cells offer a model system for studying the molecular events that control the induction of GTPCH gene expression and BH4 synthesis in glial cells.

Animals↗
PubMed1996

Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content in cultured sympathetic neurons by leukemia inhibitory factor and ciliary neurotrophic factor.

Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase. (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3-7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78-96% reduction in GTPCH mRNA content relative to beta-actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.

Adrenergic Fibers↗
PubMed1995

Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA.

6-Pyruvoyl tetrahydropterin synthase catalyzes the second step in the biosynthesis of tetrahydrobiopterin. In the present study, the reverse transcription-polymerase chain reaction technique was used to clone a portion of 6-pyruvoyl tetrahydropterin synthase cDNA from rat pineal gland RNA. The sequence of this cDNA was found to be essentially identical to that previously reported for the rat liver. 6-Pyruvoyl tetrahydropterin synthase mRNA levels in various rat tissues, including the brain, were then analyzed by Northern blot and nuclease protection assay. A single 1.35 kb transcript of 6-pyruvoyl tetrahydropterin synthase mRNA was detected by Northern blot analysis in the rat adrenal gland, brain-stem, and liver. Quantitation by nuclease protection assay demonstrated that 6-pyruvoyl tetrahydropterin synthase mRNA was most abundant in the adrenal gland, kidney, and pineal gland (19.5-25.5 amol/microgram RNA). Relatively homogeneous levels of 6-pyruvoyl tetrahydropterin synthase mRNA were found in various brain regions including the cerebellum, substantia nigra and locus coeruleus (4.12-12 amol/microgram RNA). In the adrenal gland, 6-pyruvoyl tetrahydropterin synthase and tyrosine hydroxylase mRNAs were elevated between 3 and 4-fold 24 h after a single dose of reserpine (10 mg/kg), a treatment known to increase tetrahydrobiopterin levels in this tissue. This result suggests that although 6-pyruvoyl tetrahydropterin synthase is not believed to be rate-limiting in the tetrahydrobiopterin biosynthetic pathway, control of gene expression for this enzyme may play an essential role in regulating the synthesis of this important cofactor.

5'-Nucleotidase↗
PubMed1995

Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content by nerve growth factor in cultures of superior cervical ganglia.

Monolayer cultures of superior cervical ganglia free of support cells were maintained in the presence of 100 ng/ml 7S-NGF for 4 days. The concentration of NGF was then changed to between 50 and 400 ng/ml and cultures continued for an additional 7 days. Tetrahydrobiopterin (BH4) content, GTP cyclohydrolase (GTPCH) enzyme activity and mRNA levels were then determined. All three of these measures were found to be elevated between 2- to 4-fold by treatment with increasing concentrations of NGF. Tyrosine hydroxylase (TH) enzyme activity and mRNA levels were increased from 8 to 13-fold by these same treatments. These results indicate that the content of BH4 within sympathetic neurons can be regulated by NGF receptor-mediated changes in GTPCH gene expression. Moreover, concomitant increases in TH enzyme activity and BH4 content demonstrate a coordinated regulation by NGF of this enzyme and its essential cofactor.

Analysis of Variance↗
PubMed1994

Dopamine and gamma-aminobutyric acid transporters: differential regulation by agents that promote phosphorylation.

Treatment of striatal synaptosomes with the protein phosphatase inhibitor okadaic acid significantly decreased gamma-aminobutyric acid (GABA) uptake, indicating that the GABA transporter may be regulated by phosphorylation. Forskolin and 8-bromoadenosine-3,5-cyclic monophosphate (8-br-cAMP) inhibited GABA uptake to the same extent as okadaic acid, suggesting the involvement of protein kinase A in GABA transporter regulation. In contrast, the same treatments did not alter dopamine (DA) uptake into striatal synaptosomal preparations. The results suggest that the structurally related GABA and DA transporters may be subject to different post-translational regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Regulation of tetrahydrobiopterin biosynthesis in cultured dopamine neurons by depolarization and cAMP.

Primary cultures containing embryonic rat brain mesencephalic or hypothalamic dopamine neurons were used to examine the effects of membrane depolarization and elevations of cAMP levels on tetrahydrobiopterin cofactor content. Initial studies showed that 24-h incubations with 8-bromo-cAMP or isobutyl methylxanthine increased cofactor levels in either culture system, whereas the stimulatory effects of forskolin or depolarization of membrane potential were only observed in cultures of hypothalamus. 8-Bromo-cAMP was found to increase cofactor content in a concentration-dependent manner, with increases observed up to 5 mM. The time course of the effect of 8-bromo-cAMP was biphasic. Over the short term, an increase of 50% in cofactor content at 2 and 5 h was detected. Over the long term, by 24-48 h, cofactor levels increased by between 100% and 300%. Studies of cofactor turnover indicated that the long-term increase was due to stimulation of tetrahydrobiopterin biosynthesis with no alteration in degradation rate. Inhibitors of gene transcription and translation prevented the long- but not short-term increase in cofactor content. Levels of GTP cyclohydrolase I mRNA were increased 7-10-fold following 5 h of incubation with 8-bromo-cAMP. Tetrahydrobiopterin biosynthesis within cultured dopamine neurons of the hypothalamus and mesencephalon thus appears to be regulated by a cAMP-dependent mechanism involving enhanced gene expression of enzyme(s) involved in cofactor biosynthesis.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Dopamine neuron membrane physiology: characterization of the transient outward current (IA) and demonstration of a common signal transduction pathway for IA and IK.

Dopamine neurons derived from the mesencephalon of embryonic rats were maintained in primary culture, identified and studied with whole-cell patch recording techniques. These neurons demonstrated a rapidly activating and inactivating voltage-dependent outward current which required the presence of K+ ions. This current was termed IA because of its transient nature. It was elicited by step depolarizations from holding potentials more negative than -50 mV and exhibited steady-state inactivation at a membrane potential more positive than -40 mV and half-maximal inactivation observed at -65 mV. This current rapidly achieved peak activation in less than 8 msec and decayed with a time constant (tau) of 58 +/- 5 msec. This current was observed in the presence of tetraethylammonium but was readily blocked by 4-aminopyridine (2-4 mM). This current was also observed to be modulated by stimulation of D2 dopamine receptors (DA autoreceptors) located on the dopamine neurons. Thus, both DA and the D2 receptor agonist quinpirole enhanced the peak IA observed, while the partial D1 receptor agonist SKF 38393 was without effect. The enhancement of IA was confirmed to be due to the activation of D2 receptors as the effects of either DA or quinpirole were blocked by the D2 receptor antagonists eticlopride and sulpiride, but not by the D1 receptor antagonist SCH 23390. Since we have previously demonstrated that the IK present in these cells is also enhanced by D2 receptor stimulation, we investigated the signal transduction pathways involved in coupling DA autoreceptors to both IA and IK. The response of both these potassium currents to DA autoreceptor stimulation was completely abolished by the preincubation of cultures with pertussis toxin, indicating the possible involvement of the G proteins Gi and G(o). In an attempt to further characterize which G protein may be involved, additional experiments were performed. The ability of DA autoreceptor stimulation to augment both currents was also blocked completely when G protein activation was prevented by the intracellular application of GDP beta S (100 microM). In contrast, irreversible activation of G proteins by intracellular application of the nonhydrolyzable GTP analog GTP gamma S (100 microM) mimicked the effects of DA autoreceptor stimulation on both IA and IK. In addition, the intracellular application of a polyclonal antibody that was selective for the alpha-subunit of G(o) completely abolished the DA autoreceptor modulation of both currents while preimmune serum was without effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

A comparison of the developing dopamine neuron phenotype in cultures of embryonic rat mesencephalon and hypothalamus.

Development of the dopamine (DA) neuron phenotype was monitored in cultures of embryonic rat mesencephalon (MES) and hypothalamus (HYP) maintained for 1 to 21 days in vitro (DIV) in the absence of glial support cells. Cell counts following immunohistochemistry for tyrosine hydroxylase (TH) demonstrated that the number of DA neurons declined by 85% in MES cultures yet increased 5-fold in cultures of HYP, so that by 21 DIV equal numbers of DA neurons were present in these culture systems. After 21 DIV MES DA neurons exhibited a multipolar morphology, with numerous branching processes. HYP DA neurons were primarily fusiform in shape with fewer processes and process branch points. Double-label immunohistochemistry for TH and microtubule-associated protein 2 identified the majority of TH-positive processes in either culture system as dendrites. Individual MES but not HYP DA neurons were also found to generate axons. Western analysis showed that between 1 and 21 DIV the concentration of TH protein increased 2-fold in MES and 4-fold in HYP cultures. After 21 DIV the concentration of TH protein in MES cultures was twice that found in cultures of HYP. In the period between 1 and 21 DIV levels of tetrahydrobiopterin (BH4) increased by 6-fold in MES and 20-fold in HYP cultures. After 21 DIV BH4 content was 3-fold higher in HYP than in MES cultures. The abundance of the mRNA encoding for GTP cyclohydrolase I, the rate-limiting enzyme in BH4 biosynthesis, was similar in MES and HYP cultures despite this difference in BH4 levels. In contrast, TH mRNA was 4-fold more abundant in MES than in HYP cultures. Treatment of MES cultures with the DA neuron toxin 1-methyl-4-phenylpyridinium decreased DA cell numbers, TH protein content and BH4 levels, demonstrating that BH4 is localized primarily to DA neurons. Similar treatment of HYP cultures did not effect any of these parameters. Steady-state levels of DA and the rate of DA synthesis were both 3-fold higher in MES than in HYP cultures. A 95% decline in BH4 content produced by inhibiting BH4 biosynthesis resulted in 64% and 84% declines in the rate of MES and HYP DA synthesis, respectively. Overall, these observations indicate that, with the exception of the capacity to synthesize DA, DA neurons in MES and HYP cultures share few common properties.

1-Methyl-4-phenylpyridinium↗
PubMed1993

Neurotrophic effects of substance P on hippocampal neurons in vitro.

The potential neurotrophic effect of substance P-like immunoreactivity present in culture media was assessed in rat embryonic day 18 hippocampal cultures. The neurokinin-1 (substance P) receptor antagonist CP-96345 induced neurotoxicity that was dose dependent and attenuated by addition of substance P or the neurokinin-1 agonist [Sar9,Met(O2)11]-SP. These studies suggest that under some conditions neurokinin-1 receptor stimulation promotes neuronal survival.

Animals↗
PubMed1993

Substance P gene expression in sympathetic neurons is regulated by neuron/support cell interaction.

In agreement with previous findings, the presence of support cells was found to increase the level of preprotachykinin (i.e. substance P-encoding) mRNA in cultures of sympathetic neurons. Treatment of neuron-only cultures, which did not express detectable levels of preprotachykinin mRNA, with conditioned medium from support cell-only cultures, also increased the level of preprotachykinin mRNA. This elevation in substance P gene expression was reflected in a 2-fold increase in the number of substance P-like immunoreactive neurons. In contrast, treatment of neuron-only cultures with conditioned medium from co-cultures of sympathetic neurons and support cells did not increase the level of preprotachykinin mRNA or the number of neurons containing substance P-like immunoreactivity. These observations suggest that while support cells release a soluble factor(s) capable of inducing substance P expression in sympathetic neurons, the production or action of this factor(s) is inhibited by the interaction between support cells and sympathetic neurons. Thus, by interacting with non-neuronal cells in their environment, sympathetic neurons appear to play an active role in determining which neurotransmitter phenotype they express.

Animals↗
PubMed1993

Differential metabolism of tetrahydrobiopterin in monoamine neurons: a hypothesis based upon clinical and basic research.

This chapter has attempted to describe and integrate some of the clinical and basic research that support our hypothesis that the metabolism of BH4 is normally heterogeneous across different populations of monoamine-containing neurons. Based upon this hypothesis, there may now be reason to support the idea that certain neuropsychiatric illnesses, which are though to be the result (at least in part) of altered monoamine metabolism, might find their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups. Such a specific dysfunction might not be apparent in the rest of the brain or peripheral nervous system, thereby being difficult to detect. Perhaps the application of molecular biological techniques to studies of BH4 metabolism in man will shed new light on these problems.

Animals↗
PubMed1993

GABA transporter mRNA: in vitro expression and quantitation in neonatal rat and postmortem human brain.

A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by nuclease protection assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by nuclease protection assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.

Adult↗
PubMed1993

Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridization. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1.2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2- and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.

Animals↗
PubMed1993

Transfected D2 short dopamine receptors inhibit voltage-dependent potassium current in neuroblastoma x glioma hybrid (NG108-15) cells.

Two isoforms of the D2 dopamine receptor exist, termed D2 short (D2s) and D2 long, which differ by the presence or absence of 29 amino acids. To examine the possible coupling of the D2s isoform to voltage-dependent K+ current, NG108-15 cells that were transfected with and stably express this isoform were studied using whole-cell patch-clamp techniques. In transfected, but not untransfected, cells dopamine and quinpirole (QUIN) reduced the normally observed peak outward K+ current, and this effect was abolished by the D2 antagonist sulpiride but not by the alpha 2-adrenergic receptor antagonist idazoxan or the D1 antagonist (R)-(+)-SCH-23380. The D1 receptor agonist SKF 38393 had no effect. QUIN-induced inhibition of K+ current was prevented by loading the cells with the Ca(2+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting a critical role for intracellular Ca2+ mobilization. In contrast, reduction of the concentration of extracellular Ca2+ and inclusion of the Ca2+ channel blocker cobalt did not modify the reduction of K+ current produced by stimulation of D2s receptors. A critical role for intracellular calcium mobilization in the observed effects was further supported by the observation that increases in cytosolic Ca2+ produced by thapsigargin mimicked the effect of QUIN, whereas intracellular ryanodine, which blocks Ca2+ mobilization, abolished the QUIN responsiveness. Finally, the effect of D2S activation on K+ current was not modified by pretreatment of the cells with pertussis toxin. These results suggest that the D2s dopamine receptor expressed in NG108-15 cells inhibits the activity of native K+ current via a mechanism that is dependent upon the mobilization of intracellular Ca2+ and does not involve a pertussis toxin-sensitive G protein.

Calcium↗